Inhibition of Syk kinase expression

ABSTRACT

The present invention relates, in general, to Syk kinase and, in particular, to a method of inhibiting Syk kinase expression using small interfering RNA (siRNA).

This application claims priority from Provisional Application No.60/484,299, filed Jul. 3, 2003, the entire contents of which areincorporated herein by reference.

TECHNICAL FIELD

The present invention relates, in general, to Syk kinase and, inparticular, to a method of inhibiting Syk kinase expression using smallinterfering RNA (siRNA).

BACKGROUND

Double stranded RNA has been shown to be a powerful agent forinterfering with gene expression in a number of organisms, including C.elegans and Drosophila, as well as plants (Bernstein et al, RNA7:1509-2151 (2001), McManus et al, Nat. Rev. Genet. 3:737-747 (2992),Hutvagner et al, Curr. Opin. Genet. Dev. 12:225-232 (2002), Zamore, Nat.Struct. Biol. 8:746-750 (2001) Tuschl et al, Genes Dev. 13:3191-3197(1999)). Early problems in silencing mammalian genes with doublestranded RNA arose because the mammalian immune system destroys cellsthat contain double stranded RNA, through mechanisms such as theinterferon response, evolved as defense against invading RNA viruses(Clarke et al, RNA 1:7-20 (1995)). It has been demonstrated, however,that very short RNA fragments (e.g., 20-23 nt in length), designatedsmall interfering RNA (siRNA), are able to escape the immune response.Thus introduced siRNAs can function well as gene silencing agents inmammalian cells (Elbashir et al, Nature 411:494-498 (2001), Elbashir etal, Genes Dev. 15:188-200 (2001), Paddison et al, Genes Dev. 16:948-958(2002), Wianny et al, Nat. Cell Biol. 2:70-75 (2000)).

As it is presently understood, RNAi involves a multi-step process.Double stranded RNAs are cleaved by the endonuclease Dicer to generate21-23 nucleotide fragments (siRNA). The siRNA duplex is resolved into 2single stranded RNAs, one strand being incorporated into aprotein-containing complex where it functions as guide RNA to directcleavage of the target RNA (Schwarz et al, Mol. Cell. 10:537-548 (2002),Zamore et al, Cell 101:25-33 (2000)), thus silencing a specific geneticmessage (see also Zeng et al, Proc. Natl. Acad. Sci. 100:9779 (2003)).

Anti-sense DNA has also been widely used to inhibit gene expression(Roth et al, Annu. Rev. Biomed. Eng. 1:265-297 (1999)). Once inside thecell, anti-sense oligonucleotides (ASO) recognize, then bind tightly tocomplementary mRNA, thus preventing the mRNA from interacting with theprotein translation machinery of the cell.

It has been demonstrated that inhibition of Syk kinase expression by Sykkinase mRNA ASO dramatically diminishes Fcγ receptor signaling (Matsudaet al, Molec. Biol. of the Cell 7:1095-1106 (1996)), and that Syk kinasemRNA ASO introduced by aerosol into rat lungs protects against Fcγreceptor-induced lung inflammation (Stenton et al, J. Immunol.169:1028-1036 (2002)).

At least in certain systems, siRNA is more potent and reliable than ASOas an inhibitor of gene expression. The present invention results fromstudies designed to test the efficacy of siRNA as an inhibitor of Sykkinase expression.

SUMMARY OF THE INVENTION

The present invention relates generally to Syk kinase. In a preferredembodiment, the invention relates to a method of inhibiting Syk kinaseexpression using small interfering RNA (siRNA) and to therapeuticstrategies based on such a method.

Objects and advantages of the present invention will be clear from thedescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The sense strand of each Syk kinase siRNA is the same sequenceas the target sequence with the exception of the initial templateadenine dimer and terminal overhang thymidine dimer. The antisensestrand of the siRNA is the reverse complement of the target sequence.

FIG. 2. Expression of Syk kinase in RBL-2H3 cells transfected with siRNAtargeted to Syk kinase mRNA. RBL-2H3 cells were transfected with siRNA-1(Lane 2), siRNA-2 (lane 3) or lipofectamine transfection control (Lane1). Proteins in cell lysates were separated by SDS-PAGE and transferredto nitrocellulose. Top panel, Syk kinase immunoblot; bottom panel, actinimmunoblot.

FIG. 3. Expression of Syk kinase in human monocytes transfected withsiRNA targeted to Syk kinase mRNA. Monocytes were treated with siRNA(Lane 2) or lipofectamine transfection control (Lane 1). Proteins incell lysates were separated by SDS-PAGE, transferred to nitrocelluloseand immunoblotted with anti-Syk kinase antibody.

FIG. 4. Western blot analysis: Syk protein expression in HS-24 cells.

FIGS. 5A and 5B. (FIG. 5A) HS-24 cells, following siRNA treatment, werelysed and equal amounts of total protein in HS-24 cell lysates wereresolved by 10% SDS gel electrophoresis, and analyzed by Western blotusing monoclonal antibody to Syk or actin. Lane 1—no treatment, lane2—siRNA-1 (control) 24 h treatment, lane 3—siRNA-1 48 h treatment, lane4—siRNA-2 24 h treatment, lane 5—siRNA-2 48 h treatment. (FIG. 5B) RNAwas isolated and RT-PCR was performed for Syk and β-actin. Lane 1—notreatment, lane 2—siRNA-1 (control) 48 h treatment, lane 3—siRNA-2 48 htreatment.

FIGS. 6A and 6B. HS-24 cells plated on either polylysine coated plates(non stimulated, resting) or fibronectin coated plates (stimulated) weretreated with siRNA-2, or siRNA-1 (control), or piceatannol. Cells weretreated with 10 ng/ml of TNF during overnight culture. (FIG. 6A)Following siRNA (48 h) or piceatannol (16 h) treatment, cells wereremoved, immunostained with anti-CD54 (ICAM-1) and analysed by flowcytometry. (FIG. 6B) Cell culture supernatants were analysed for IL-6release using an IL-6 ELISA kit. *P<0.05, **P<0.005 as compared tountreated cells (e.g., untreated with siRNA) stimulated with TNF.Results are representative of three to five independent experiments. Thedata indicate that inhibition of Syk expression by siRNA-2down-regulates TNF-induced ICAM-1 expression and IL-6 release, importantin the inflammatory response.

FIGS. 7A and 7B. Effect of siRNA targeted to Syk kinase delivered viaaerosol on total cell numbers in bronchoalveolar lavage (BAL) fluid ofovalbumin (OA) sensitized and challenged Brown Norway Rats after threetreatments. (FIG. 7A provides data as bar graphs, FIG. 7B showsindividual data points (individual animals).

FIGS. 8A-8D. Effect of siRNA targeted to Syk kinase delivered viaaerosol on numbers of macrophages, neutrophils, lymphocytes andeosinophils in BAL fluid of OA sensitized and challenged Brown Norwayrats after three treatments. (FIG. 8A provides data as bar graphs, FIG.8B shows individual data points (individual animals) for macrophagenumbers, FIG. 8C shows individual data points for neutrophil numbers,and FIG. 8D shows individual data points for eosinophil numbers.)

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to RNA molecules that target Syk kinasemRNA. For example, the invention relates to RNA molecules about 19, 20or 21 to about 23 nucleotides in length that direct cleavage and/ordegradation of Syk kinase mRNA.

In a preferred embodiment, the present invention relates to the use ofsiRNA molecules, double stranded RNA molecules typically comprising two20-23 nucleotide (nt) strands. SiRNAs suitable for use in the inventioncan be produced using any of a variety of approaches. The siRNA can beprepared in vitro and then introduced directly into cells (for example,by transfection). Alternatively, intracellular expression can beeffected by transfecting into cells constructs (e.g., DNA-based vectorsor cassettes) that express siRNA within cells.

More specifically, siRNA suitable for use in the invention can beprepared, for example, via chemical synthesis, in vitro transcription,enzymatic digestion of a longer dsRNA using an RNase III enzyme such asDicer or RNase III, expression in cells from an siRNA expression plasmidor viral vector, or expression in cells from a PCR-derived siRNAexpression cassette. Detailed descriptions of these various approachesare readily available and can be found, for example, athttp://www.ambion.com/techlib/tn/103/2.html, www.bdbiosciences.com,www.oligoengine.com, www.genetherapysystems.com, www.dharmacon.com,http://www.mpibpc.gwdg.de/abteilungen/100/105/sirna.html, and/or in thereferences cited therein (which references are also incorporated hereinby reference). (See also Sui et al, Proc Natl Acad Sci USA 99: 5515-20(2002), Brummelkamp et al, Science 296:550-3 (2002),Paul et al, NatureBiotechnology 20:505-8 (2002), Lee et al, Nature Biotechnology 20: 500-5(2002), Castanotto et al, RNA 8: 1454-60 (2002) and US Appln.20030108923.)

Various approaches are available to enhance stability of RNA of theinvention, (see, for example, U.S. Application Nos. 20020086356,20020177570 and 20020055162, and U.S. Pat. Nos. 6,197,944, 6,590,093,6,399,307, 6,057,134, 5,939,262, and 5,256,555, and references citedtherein).

As indicated above, siRNA suitable for use in the invention can beprepared chemically. Advantageously, 2′ hydroxyls are protected duringthe synthetic process against degradation using, for example, acidlabile orthoester protecting groups (see Scaringe et al, J. Am. Chem.Soc. 120:11820 (1998) and www.dharmacon.com (e.g., the ACE technologydescribed therein)). The RNA oligomers can be simultaneously 2′deprotected and annealed prior to use.

In chemically synthesized siRNA, at least one strand of the doublestranded molecule can have a 3′ overhang from about 1 to about 6nucleotides (e.g., pyrimidine and/or purine nucleotides) in length.Preferably, the 3′ overhang is from about 1 to about 5 nucleotides(e.g., thymidines or uridines), more preferably from about 1 to about 4nucleotides and most preferably 2 or 3 nucleotides in length.Advantageously, each strand has an overhang. The length of the overhangscan be the same or different for each strand. Typically, both strandshave overhangs of the same length. In a particular embodiment, the RNAof the present invention comprises 21 or 22 nucleotide strands that arepaired and that have overhangs of from about 1 to about 3, particularly,about 2, nucleotides on the 3′ ends of both of the RNA strands.

As indicated above, siRNAs suitable for use in the invention can beprepared by enzymatic digestion of a longer dsRNA using an RNase IIItype enzyme (e.g., Dicer). (See references and web sites cited above.)For example, a commercially available Dicer siRNA generation kit can beused that permits generation of large numbers of siRNAs from full lengthtarget genes (Gene Therapy Systems, Inc, MV062603). SiRNA can beproduced from target DNA and T7 RNA polymerase promoter sequences usingPCR based cloning. Following RNA transcription from the target sequence,recombinant Dicer can cleave the transcribed RNAi into 22 bp siRNAs.

Also as indicated above, siRNA molecules suitable for use in the presentinvention can also be recombinantly produced using methods known in theart. (See references and web sites cited above.) Recombinant technologypermits in vivo transcription of siRNAs in mammalian cells. Inaccordance with this approach, vectors can be used that contain, forexample, RNA polymerase III or U6 promoter sequences. Such vectors(including viral vectors and plasmid vectors (such as pSIREN)) can beused as expression vectors or as shuttle vectors in conjunction withviral systems (e.g., adenoviral or retroviral systems) to introducesiRNA into mammalian cells. Vectors can be engineered to express senseand anti-sense strands of siRNAs that anneal in vivo to producefunctional siRNAs. Alternatively, hairpin RNA can be expressed byinserting into a vector the sense strand (e.g., about 20 nt) of thetarget, followed by a short spacer (e.g., about 4 to about 10 nt), thenthe antisense strand of the target (e.g., about 20 nt) and, for example,about 5-6 T's as transcription terminator. The resulting RNA transcriptfolds back to form a stem-loop structure comprising, for example, abouta 20 bp stem and about a 10 nt loop with 2-3 U's at the 3′ end. (Seealso Paddison et al (Proc. Natl. Acad. Sci. 99:1443-1448 (2002).)Constructs suitable for use in effecting in vivo production (includingselection of vectors and promoters) can be readily designed by oneskilled in the art and will vary, for example, with the cell/tissuetarget and the effect sought.

dsRNA can be used in the methods of the present invention provided ithas sufficient homology to the targeted Syk kinase mRNA. SiRNA duplexescan be designed, for example, by searching Syk kinase cDNA for thetarget motif “AA(N)₁₉”, wherein N is any nucleotide, motifs withapproximately 30% to 70% G/C content being preferred, those of about 50%G/C content being more preferred. The sense strand of the siRNA duplexcan correspond to nucleotides 3 to 21 of the selected AA(N)₁₉ motif. Theantisense strand of the siRNA duplex can have a sequence complementaryto nucleotides 1 to 21 of the selected AA(N)₁₉ motif. Further designdetails are provided athttp://www.mpibpc.gwdg.de/abteilungen/100/105/sirna.html.

Preferred target sequences include sequences unique to Syk kinase mRNA.For example, target sequences can be selected from sequences between thetwo SH2 domains of Syk kinase or between the second SH2 domain and thekinase domain. Certain specific DNA target sequences are described inthe non-limiting Examples that follow. Additional targets include, butare not limited to, the following: Identified homologies of *Sequence %GC 16-18/19 nucleotides AATATGTGAAGCAGACATGGA 42 mitochondrial ribosomalprot15 AATCAAATCATACTCCTTCCC 42 AAGAGAGTACTGTGTCATTCA 42AAGGAAAACCTCATCAGGGAA 47 inositolhexaphosphate kinase, β globin on Chr11AATCATACTCCTTCCCAAAGC 47 AATTTTGGAGGCCGTCCACAA 53 oxytokinaseAAGACTGGGCCCTTTGAGGAT 58 AAGCACACATGGAACCTGCAG 58 histamine receptor H3,GTP binding protein AACTTCCAGGTTCCCATCCTG 58 AAGCCTGGCCACAGAAAGTCC 63AAGCCCTACCCATGGACACAG 63 AACCTGCAGGGTCAGGCTCTG 68 AAGGGGTGCAGCCCAAGACTG68 γ glutamyl transferase, rb prot L27a AACTTGCACCCTGGGCTGCAG 68 calciumchannel α1E subunit AAGTCCTCCCCTGCCCAAGGG 74 NADH; ubiquinone oxido-reductase MLRQ subunit AAGGCCCCCAGAGAGAAGCCC 74 AATCTCAAGAATCAAATCATA 26AATGTTAATTTTGGAGGCCGT 42 AATCCGTATGAGCCAGAACTT 47 AATCGGCACACAGGGAAATGT53 AACCGGCAAGAGAGTACTGTG 58 AAGGAGGTTTACCTGGACCGA 58

The siRNAs described herein can be used in a variety of ways. Forexample, the siRNA molecules can be used to target Syk kinase mRNA in acell or organism. In a specific embodiment, the siRNA can be introducedinto human cells or a human in order to mediate RNAi in the cells or incells in the individual, so as to prevent or treat a disease orundesirable condition associated with Syk kinase expression (e.g.,inflammation of the lungs, joints eyes or bladder). The siRNA can alsobe used in the treatment of the immune destruction of blood cells, e.g.,red blood cells in autoimmune hemolytic anemia and platelets in immunethrombocytopenic purpurea (ITP) (e.g., by targeting Syk kinase mRNA inmacrophages and spleen and liver cells). In accordance with the instantmethod, the Syk kinase gene is targeted and the corresponding mRNA (thetranscriptional product of the targeted Syk kinase gene) is degraded byRNAi. When lung cells are the target, an siRNA-containing compositioncan be aerosolized and administered, for example, via inhalation.Administration to joints can be effected by injection of ansiRNA-containing solution. Administration to the eyes can be effected,for example, by injection or by application of drops comprising thesiRNA in a carrier. Administration to the bladder, etc. can be effected,for example, by washing or irrigating the target tissue with acomposition containing the siRNA. Administration to the skin can be viatopical administration (e.g., as a liquid, cream or gel).

In accordance with the invention, cells of an individual (e.g., bloodmononuclear cells, basophiles or mast cells) can be treated ex vivo soas to effect degradation of the Syk kinase mRNA. The cells to be treatedcan be obtained from the individual using known methods and the siRNAsthat mediate degradation of the corresponding Syk kinase mRNA can beintroduced into the cells, which can then be re-introduced into theindividual.

In a specific embodiment, the invention relates to the use of theabove-described siRNAs to inhibit mediator (e.g., histamine) releasefrom cells bearing an Fcε receptor, such as mast cells. Inhibition ofhistamine (a mast cell mediator) release, for example, is of therapeuticimportance in the treatment of asthma.

The siRNAs (or constructs suitable for use in effecting intracellularproduction of siRNA) of the invention can be administered systemically(e.g., via IV) or directly to the target tissue (e.g., via aerosoladministration to the lung). Delivery can be effected using thetechniques described herein (including liposome formulations). Inaddition to liposome formulations, polymer formulations can be used.Polyethylenimine (PEI) is an example of a suitable cationic polymer.Varying sizes of PEI can be used, including linear 22 kDa and branched25 kDa PEI (other sizes, modified and unmodified, as well asbiodegradable versions can be used). Delivery can also be effectedusing, for example, non-toxic viral delivery systems (e.g., anadeno-associated viral delivery system). Optimum dosing will depend onthe patient, the siRNA, the mode of administration, and the effectsought. Optimum conditions can be established by one skilled in the artwithout undue experimentation.

Certain aspects of the invention can be described in greater detail inthe non-limiting Examples that follows. (See also US Applications20030084471, 20030108923 and 20020086356.)

EXAMPLE 1

Experimental Details

Reagents

Lipofectamine 2000 and Opti-Mem were purchased from Invitrogen (SanDiego, Calif.). Eagle's MEM (EMEM), FCS, penicillin, and streptomycinwere purchased from Life Technologies (Grand Island, N.Y.). Rabbitanti-murine Syk kinase polyclonal antibodies (Ab) and anti-actin Ab werepurchased from Santa Cruz Biotechnology (Santa Cruz, Calif.), andF(ab′)₂ goat anti-rabbit Ab was supplied by The Jackson Laboratory (BarHarbor, Me.). Chemiluminescence reagent was purchased from DuPont NEN(Boston, Mass.).

Cells and Cell Lines

Peripheral blood monocytes obtained from healthy volunteers at theUniversity of Pennsylvania were isolated as previously described(Matsuda et al, Molec. Biol. of the Cell 7:1095-1106 (1996)). Briefly,heparinized blood was centrifuged on Ficoll-Hypaque (LymphocyteSeparation Medium; Organon Teknika, Durham, N.C.) and interface cellswere resuspended in complete medium containing RPMI 1640 (GIBCO BRL,Grand Island, N.Y.), 10% heat inactivated-fetal calf serum (FCS)(Intergen, Purchase, N.Y.) and 2 mM L-glutamine. Cells were allowed toadhere at 37° C. for 30 min to tissue culture flasks precoated with FCS.After 45-90 min, nonadherent cells were removed by extensive washing inHanks' balanced salt solution. Cells were harvested by vigorousagitation. Monocytes were routinely>98% viable as judged by Trypan Blueexclusion. Isolated monocytes were maintained in RPMI 1640 supplementedwith L-glutamine (2 mM) and 10% heat inactivated FCS at 37° C. in 5%CO₂.

Rat basophilic cells (RBL-2H3) were grown in EMEM supplemented with 17%FBS, 100 U penicillin, 100 μg/ml streptomycin, and 4 mM glutamine at 37°C. in 5% CO₂.

siRNA Duplex Construction

siRNAs for Syk kinase were prepared by Dharmacon Research Inc.(Lafayette, Colo.). In designing the siRNAs according to the guidelinesprovided by the manufacturer, potential siRNA targets (19 nucleotidesimmediately downstream of AA pairs) in human Syk kinase RNA were firstidentified. These sites were then scanned in the sequences of rat andmouse Syk kinase RNA in order to identify common Syk kinase RNA targetsequences in these species. Two appropriate sites were identified, andtwo 21-mer RNAs, each consisting of 19 complementary nucleotides and 3′terminal noncomplementary dimers of thymidine (Elbashir et al, Nature411:494-498 (2001)), were constructed. The sense strand of the siRNA isthe same sequence as the target mRNA sequence with the exception of theterminal thymidine dimer. The antisense strand of the siRNA is thereverse complement of the target sequence. 1) siRNA-1: Human, bp 296 tobp 316; Mouse and Rat, bp 307 to bp 327. sense5′-     gaagcccuucaaccggccc dTdT 3′ antisense 3′-dTdTcuucgggaaguuggccggg 5′ 2) siRNA-2: Human, bp 364 to bp 382; Mouse andRat, bp 375 to bp 393 sense 5′-     ccucaucagggaauaucug dTdT 3′antisense 3′-dTdT ggaguagucccuuauagac 5′Transfections

siRNA was introduced into RBL-2H3 cells and into monocytes bytransfection. For the transfections, 5×10⁴ RBL-2H3 cells or 1×10⁵monocytes were seeded into each well of a 24-well plate. Twenty-fourhours later, the complete medium was replaced with 400 μl fresh mediumlacking serum and antibiotic and siRNA/Lipofectamine 2000 complex wasadded to each well. For the RBL cells, the siRNA/Lipofectamine 2000complex was formed by adding 3 μl of siRNA duplex (20 μM) and 3 μl ofLipofectamine 2000 to 100 μl Opti-mem without serum or antibioticaccording to the manufacturer's protocol. For monocytes, thesiRNA/Lipofectamine 2000 complex was formed by adding 3 μl of siRNAduplex (20 μM) and 1 μl of Lipofectamine 2000 to 100 μl Opti-mem withoutserum or antibiotic. Cells were incubated at 37° C. for 48 hours beforeexamination of kinase protein expression by Western blotting.

Western Blot Analysis of Syk Kinase Protein

Lysates were prepared by boiling cells in Laemmli sample buffer (2% SDS,10% glycerol, 100 mM DTT, and 60 mM Tris (pH 6.8) for 5 minutes.Proteins in lysates were separated by SDS-PAGE (10% polyacrylamide) andtransferred to a nitrocellulose membrane in sample buffer (25 mM Tris,190 mM glycine, and 20% methanol). The nitrocellulose membrane wasincubated overnight at 4° C. with 1 μg/ml rabbit anti-murine Syk kinsaepolyclonal Ab before incubation with goat anti-rabbit HRP (1.5 h at roomtemperature). Protein bands on the membrane were visualized withchemiluminescence reagent according to the manufacturer's protocol.After detection of Syk kinase protein, the anti-Syk kinase Ab wasremoved by incubating the membrane in a stripping buffer containing 100mM 2-ME, 2% SDS, and 62.5 mM Tris-HCL (pH 6.7) for 30 minutes at 50° C.with occasional agitation. The membrane was then reprobed withanti-actin Ab and bands on the membrane were visualized withchemiluminescence reagent. Protein levels of Syk kinase were quantifiedby densitometry (Personal Densitometer, Molecular Dynamics).

Results

Effects of siRNA on the Expression of Syk Kinase

Rat basophilic cells (RBL-2H3 cells) and human monocytes weretransfected with siRNA directed to sequences common to rat, mouse andhuman Syk kinase RNA. The expression of Syk kinase protein was analyzedby Western blotting using anti-Syk kinase Ab (FIGS. 2 and 3). Inhibitionof Syk kinase expression in RBL cells treated with siRNA is shown inFIG. 2, and levels of Syk kinase protein, normalized to levels of actinprotein, are presented in Table 1. Actin is a common protein used as astandard to examine protein expression. Syk kinase protein expression insiRNA-treated RBL cells was inhibited by 45-51% (FIG. 2, lanes 2 and 3)compared to untreated RBL cells (lane 1). The magnitude of theinhibition of Syk kinase gene expression by siRNA in cultured cells wasencouraging since previous experiments with Syk kinase mRNA ASOindicated that greater levels of Syk kinase inhibition can be achievedin non-multiplying cells such as monocytes than in multiplying cellscultured in vitro. Inhibition of Syk kinase expression by siRNA inmonocytes maintained in culture (FIG. 3) was also observed. Syk kinaseexpression in siRNA-treated monocytes was inhibited (FIG. 3, lane 2). Inaddition, Syk kinase expression in siRNA-treated U937 cells andsiRNA-treated THP-1 cells was inhibited (U937 and THP-1 beingmacrophage-like cell lines). The data thus demonstrate the effectivenessof siRNA directed against Syk kinase RNA to suppress the expression ofthis gene and indicate that siRNA directed against Syk kinase RNA canserve as a powerful therapeutic tool to combat inflammation. TABLE 1Densitometry quantification of Syk kinase expression in RBL cells.Transfection Syk kinase Syk kinase Control siRNA1 siRNA2 Syk* 2040 953627 Syk Corr** — 1114 997 % Syk Inhibition 0 45 51*densitometry units**Syk densitometry units corrected for % actin expression in each lane.Discussion

The siRNAs used in these studies were chemically synthesized (DharmiconResearch Inc., Lafayette, Colo.) but siRNAs can also be prepared byrecombinant techniques. siRNA duplexes can comprise 21-nucleotide senseand 21-nucleotide antisense strands, paired in a manner to have a2-nucleotide (dT) 3′ overhang.

The targeted region for siRNAs can be the sequence AA(N19) (N, anynucleotide) selected from the designated cDNA sequence beginning 50 to100 nucleotides downstream of the start codon. G/C-contents ofapproximately 50% are preferred. Since expression of RNAs from pol IIIpromoters is only efficient when the first transcribed nucleotide is apurine, it is preferred that the sense and antisense siRNAs begin with apurine nucleotide so that they can be expressed from pol III expressionvectors without a change in targeting site.

In the studies described above, potential siRNA targets in human Sykkinase mRNA were selected and then the sequences in rat and mouse Sykkinase mRNA were scanned in order to identify common Syk kinase mRNAtarget sequences in these species. Two appropriate target sequences wereidentified: Targeted region (1) (cDNA): 5′aagaagcccttcaaccggccc;Targeted region (2) (cDNA): 5′aacctcatcagggaatatctg.

The target sequences and Syk kinase siRNAs are shown in FIG. 1. DuplexedRNA is not highly susceptible to nuclease degradation and the use ofdeoxynucleotides (thymidine (T) rather than uridine (U)) may affect thestability of the 3′ overhang.

EXAMPLE 2

HS-24 cells (2 × 10⁵) were pre-treated with 3 μl of siRNA-1 (control)DNA Target: 5′ AAGAAGCCCTTCAACCGGCCC Sense siRNA5′-     gaagcccuucaaccggccc dTdT 3′ Antisense 3′-dTdTcuucgggaaguuggccggg 5′ siRNA or siRNA-2 DNA Target:5′ AACCTCATCAGGGAATATCTG sense 5′-     ccucaucagggaauaucug dTdT 3′antisense 3′-dTdT ggaguagucccuuauagac 5′or Syk antisense, with Lipofectamine 2000 in a 12-well plate for 24 or48 hr, and stimulated by 10 ng/ml of TNF overnight. Read-outs: IL-6 inculture supernatant (ELISA) and cell surface expression of ICAM-1 (Flowcytometry). As shown by Western blot analysis (see FIG. 4), siRNA-2caused a decrease in Syk protein expression following 48 hr oftreatment.

More specifically, HS-24 cells were transiently transfected with siRNA-2or siRNA-1 (control). Cell surface expression of ICAM-1, as well asrelease of IL-6, were then examined. Forty-eight-hour treatment of HS-24cells with siRNA-2, but not with siRNA-1, significantly suppressed bothSyk protein (FIG. 5A) and mRNA (FIG. 5B) expression.

HS-24 cells constitutively expressed low levels of ICAM-1 (FIG. 6A) notaffected by siRNA-2 treatment (not shown). Stimulation of HS-24 cellswith 10 ng/ml of TNF during overnight culture caused significantincrease in ICAM-1 expression both in resting cells (plated onpoly-L-lysine) and cells adherent to fibronectin (FIG. 6A). Cellsadherent to fibronectin showed higher expression of ICAM-1 followingstimulation with TNF compared to cells adherent to poly-L-lysine(P<0.05). Transfection with siRNA-2 down-regulated TNF-induced ICAM-1expression in fibronectin-plated HS-24 cells (P<0.005), but had nosignificant effect on ICAM-1 in poly-L-lysine-plated cells. Overnighttreatment with the pharmacological Syk inhibitor, piceatannol (10 μM),caused significant down-regulation of ICAM-1 in TNF-stimulated cellsboth in poly-L-lysine and fibronectin adherent conditions (FIG. 6A). Asdetermined by trypan blue dye exclusion, the treatment of cells withsiRNA or piceatannol had no significant effect on viability (in allexperiments, viability was >96%).

Although IL-6 release by HS-24 cells without TNF stimulation wasminimal, there was a trend to higher IL-6 levels in culture supernatantsof cells adherent to fibronectin (FIG. 6B). As expected, a greatelevation of IL-6 levels was observed following TNF stimulation in bothculture conditions, with higher levels in fibronectin-adherent cells(P<0.05). siRNA-2 treatment caused down-regulation of IL-6 release(55-58%) that reached statistical significance in fibronectin-adherentculture (P<0.05). Piceatannol almost completely inhibited TNF-inducedIL-6 release (FIG. 6B).

Thus, inhibition of Syk kinase down-regulated TNF-induced expression ofICAM-1 and IL-6 release, hallmarks of inflammatory responses in theairway epithelium. The effect was significant in cells adherent tofibronectin indicating that Syk involvement in these pro-inflammatoryevents is at least partly β1 integrin dependent.

EXAMPLE 3

The effects of siRNA targeted to Syk kinase was studied in vivo in aBrown Norway rat model of ovalbumin (OA)-induced asthma.

Brown Norway rats were sensitized to OA i.p. as described (Laberge etal, Am. J. Respir. Crit. Care Med. 151:822 (1995)) and used on day 21following sensitization.

The siRNA used in this Example is as follows: DNA Target:5′ AACCTCATCAGGGAATATCTG sense 5′-   ccucaucagggaauaucug uu 3′ antisense3′-uu ggaguagucccuuauagac    5′The description siRNA 2M, used in FIGS. 7 and 8, refers to the above,modified by Dharmacon to provide additional stability. The designationsiRNA-2, used in FIGS. 7 and 8, refers to the above sequence inunmodified form.

siRNA was used alone without liposome or was used after formation ofsiRNA/liposome complexes. 1,2 Dioleoyl-3-trimethylammonium-propane(DOTAP)/dioleoyl-phosphatidyl-ethanol-amine (DOPE) liposomes wereprepared as previously described (see Stenton et al, J. Immunol.169:1028 (2002) and references cited therein). Cationic DOTAP:DOPEliposomes were incubated at a 2.5:1 ratio of the liposome to the siRNAand 125 micrograms of siRNA (with or without liposomes) was administeredby aerosol following nebulization.

The aerosolized administration of Syk kinase-targeted siRNA was asdescribed by Stenton et al, J. Immunol. 164:3790 (2000). Nine millitersof saline, siRNA or siRNA/liposome were administered per rat bynebulization for 45 min using a Sidestream nebulizer as described inStenton et al, J. Immunol. 169:1028 (2002). Twenty-four hours later, theprocedure was repeated, followed by a third treatment at 48 h.Immediately after the third treatment, rats were challenged withaerosolized saline or 5% OA in saline for 5 min. Twenty-four hours afterchallenge, the animals were sacrificed.

Bronchoalveolar lavage (BAL) was carried out as described by Stenton etal, J. Immunol. 169:1028 (2002).

The isolated BAL cells were counted and cell smears were prepared byCytospin. Cell differentials were counted in a blinded fashion followingstaining with HEMA-3 reagent (Biochemical Sciences, Swedesboro, N.J.).

Summarizing, the Brown Norway rat model of OA induced allergic asthmaand pulmonary inflammation was used in the above-described studies.Pulmonary inflammation, as determined by recruitment of cells to BALfluid, was dramatically inhibited by Syk kinase-targeted siRNA in thepresence and absence of liposomes.

All documents and other information sources cited above are herebyincorporated in their entirety by reference.

1. A method of inhibiting expression of Syk kinase in a cell comprisingintroducing into said cell small interfering RNA (siRNA) molecules thatdirect cleavage of a target Syk kinase mRNA sequence present in saidcell thereby effecting said inhibition. 2-18. (canceled)
 19. An siRNAmolecule comprising a sequence complementary to a sequence selected fromthe group consisting of: AATATGTGAAGCAGACATGGA, AATCAAATCATACTCCTTCCC,AAGAGAGTACTGTGTCATTCA, AAGGAAAACCTCATCAGGGAA, AATCATACTCCTTCCCAAAGC,AATTTTGGAGGCCGTCCACAA, AAGACTGGGCCCTTTGAGGAT, AAGCAGACATGGAACCTGCAG,AACTTCCAGGTTCCCATCCTG, AAGCCTGGCCACAGAAAGTCC, AAGCCCTACCCATGGACACAG,AACCTGCAGGGTCAGGCTCTG, AAGGGGTGCAGCCCAAGACTG, AACTTGCACCCTGGGCTGCAG,AAGTCCTCCCCTGCCCAAGGG, AAGGCCCCCAGAGAGAAGCCC, AATCTCAAGAATCAAATCATA,AATGTTAATTTTGGAGGCCGT, AATCCGTATGAGCCAGAACTT, AATCGGCACACAGGGAAATGT,AACCGGCAAGAGAGTACTGTG, AAGGAGGTTTACCTGGACCGA, and AACCTCATCAGGGAATATCTG.


20. A composition comprising the siRNA molecule according to claim 19and a carrier.
 21. A composition comprising the siRNA molecule accordingto claim 19 and a liposome or polymer.